Linda J. Valentijn

Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes

Neuroblastoma is a childhood tumour of the peripheral sympathetic nervous system. The pathogenesis has for a long time been quite enigmatic, as only very few gene defects were identified in this often lethal tumour. Frequently detected gene alterations are limited to MYCN amplification (20%) and ALK activations (7%). Here we present a whole-genome sequence analysis of 87 neuroblastoma of all stages. Few recurrent amino-acid-changing mutations were found. In contrast, analysis of structural defects identified a local shredding of chromosomes, known as chromothripsis, in 18% of high-stage neuroblastoma. These tumours are associated with a poor outcome. Structural alterations recurrently affected ODZ3, PTPRD and CSMD1, which are involved in neuronal growth cone stabilization. In addition, ATRX, TIAM1 and a series of regulators of the Rac/Rho pathway were mutated, further implicating defects in neuritogenesis in neuroblastoma. Most tumours with defects in these genes were aggressive high-stage neuroblastomas, but did not carry MYCN amplifications. The genomic landscape of neuroblastoma therefore reveals two novel molecular defects, chromothripsis and neuritogenesis gene alterations, which frequently occur in high-risk tumours.

N‐myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis

The myc oncogenes are frequently activated in human tumors, but there is no comprehensive insight into the target genes and downstream cellular pathways of these transcription factors. We applied serial analysis of gene expression (SAGE) to identify targets of N‐myc in neuroblastomas. Analysis of 42 000 mRNA transcript tags in SAGE libraries of N‐myc‐ transfected and control neuroblastoma cells revealed 114 up‐regulated genes. The majority of these genes have a role in ribosome assembly and activity. Northern blot analysis confirmed up‐regulation of all tested transcripts. Induction was complete within 4 h after N‐myc expression. The large majority of the ribosomal proteins were induced, as well as genes controlling rRNA maturation. Cellular rRNA content was 45% induced. SAGE libraries and northern blot analysis confirmed up‐regulation of many of these genes in N‐myc‐amplified neuroblastomas. As N‐myc can functionally replace c‐myc, we analyzed whether N‐myc targets were induced by c‐myc as well. Approximately 40% of these N‐myc targets were up‐regulated in a c‐myc‐transfected melanoma cell line. These data suggest that myc genes function as major regulators of the protein synthesis machinery.

LIN28B induces neuroblastoma and enhances MYCN levels via let-7 suppression

LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B–let-7–MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives.

De-novo mutation in hereditary motor and sensory neuropathy type I

Isolated cases of hereditary motor and sensory neuropathy type I (HMSN I, Charcot-Marie-Tooth disease type 1) have been thought to be most frequently autosomal recessive. We have found that a recently discovered duplication in chromosome 17, responsible for most cases of autosomal dominant HMSN I, is present as a de-novo mutation in 9 out of 10 sporadic patients. This finding has important implications for genetic counselling of isolated patients with HMSN I.

Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells

Two genes have a synthetically lethal relationship when the silencing or inhibiting of 1 gene is only lethal in the context of a mutation or activation of the second gene. This situation offers an attractive therapeutic strategy, as inhibition of such a gene will only trigger cell death in tumor cells with an activated second oncogene but spare normal cells without activation of the second oncogene. Here we present evidence that CDK2 is synthetically lethal to neuroblastoma cells with MYCN amplification and over-expression. Neuroblastomas are childhood tumors with an often lethal outcome. Twenty percent of the tumors have MYCN amplification, and these tumors are ultimately refractory to any therapy. Targeted silencing of CDK2 by 3 RNA interference techniques induced apoptosis in MYCN-amplified neuroblastoma cell lines, but not in MYCN single copy cells. Silencing of MYCN abrogated this apoptotic response in MYCN-amplified cells. Inversely, silencing of CDK2 in MYCN single copy cells did not trigger apoptosis, unless a MYCN transgene was activated. The MYCN induced apoptosis after CDK2 silencing was accompanied by nuclear stabilization of P53, and mRNA profiling showed up-regulation of P53 target genes. Silencing of P53 rescued the cells from MYCN-driven apoptosis. The synthetic lethality of CDK2 silencing in MYCN activated neuroblastoma cells can also be triggered by inhibition of CDK2 with a small molecule drug. Treatment of neuroblastoma cells with roscovitine, a CDK inhibitor, at clinically achievable concentrations induced MYCN-dependent apoptosis. The synthetically lethal relationship between CDK2 and MYCN indicates CDK2 inhibitors as potential MYCN-selective cancer therapeutics.

Functional MYCN signature predicts outcome of neuroblastoma irrespective of MYCN amplification

Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization.

TERT rearrangements are frequent in neuroblastoma and identify aggressive tumors

Whole-genome sequencing detected structural rearrangements of TERT in 17 of 75 high-stage neuroblastomas, with five cases resulting from chromothripsis. Rearrangements were associated with increased TERT expression and targeted regions immediately up- and downstream of TERT, positioning a super-enhancer close to the breakpoints in seven cases. TERT rearrangements (23%), ATRX deletions (11%) and MYCN amplifications (37%) identify three almost non-overlapping groups of high-stage neuroblastoma, each associated with very poor prognosis.

Inhibition of a New Differentiation Pathway in Neuroblastoma by Copy Number Defects of N-myc, Cdc42, and nm23 Genes

The best studied oncogenic mechanisms are inactivating defects in both alleles of tumor suppressor genes and activating mutations in oncogenes. Chromosomal gains and losses are frequent in human tumors, but for many regions, like 1p36 and 17q in neuroblastoma, no mutated tumor suppressor genes or oncogenes were identified. Amplification of N-myc in neuroblastoma is strongly correlated with loss of 1p36 and gain of 17q. Here we report that N-myc down-regulates the mRNA expression of many genes with a role in cell architecture. One of them is the 1p36 gene Cdc42. Restoring the Cdc42 expression in neuroblastoma cells strongly induced differentiation. N-myc also inhibited Cdc42 functioning at the protein level. This was mediated by nm23-H1 and nm23-H2, which are located in the amplified 17q region. Nm23-H1 and nm23-H2 are strongly up-regulated downstream targets of N-myc. Nm23-H1 was shown to bind Cdc42 and prevented the induction of differentiation. Overexpression of Nm23 due to gain of 17q and induction by N-myc combined with weak expression of Cdc42 due to loss of 1p36 and down-regulation by N-myc can thus block differentiation. Although this marks Cdc42 as a candidate tumor suppressor gene, no mutations were found. Further silencing of Cdc42 by small interfering RNA induced massive apoptosis, indicating that tumor cell survival requires a minimal Cdc42 activity. Three regions of chromosomal gain and loss thus affect genes functioning in one pathway in neuroblastoma. They converge to bring the pathway out of balance and prevent Cdc42 mediated differentiation.

Allelic heterogeneity in hereditary motor and sensory neuropathy type la (Charcot‐Marie‐Tooth disease type 1a)

The most frequently found mutation in autosomal dominant hereditary motor and sensory neuropathy type I (HMSN I) is a large duplication on chromosome 17p11.2 containing probes VAW409R3, VAW412R3, and EW401. We investigated a family with severe features of HMSN I, and demonstrated the absence of this duplication by a quantitative analysis of the hybridization signals of VAW409R3 and VAW412R3. Linkage analysis, however, revealed linkage with probe VAW409R3a (lod score, 3.22), which demonstrates the existence of allelic heterogeneity within the HMSN la locus. These findings have implications for clinical practice and for investigating the identity of the HMSN Ia gene.

Dickkopf-3 expression is a marker for neuroblastic tumor maturation and is down-regulated by MYCN

Neuroblastoma and ganglioneuroma are neuroblastic tumors originating from the developing sympathetic peripheral nervous system. Ganglioneuromas are usually benign, while neuroblastomas have a variable prognosis and include very aggressive tumors. Examples exist of neuroblastomas regressing to ganglioneuromas and ganglioneuromas progressing to neuroblastomas. Little is known of the molecular differences between the tumor types. Here we report that Dickkopf‐3 (DKK3), a putative extra cellular inhibitor of the Wnt/β‐catenin pathway, showed a strongly differential expression between neuroblastoma and ganglioneuroma. Microarray analyses of 109 neuroblastic tumors revealed that DKK3 is strongly expressed in ganglioneuroma but only weakly in neuroblastoma. Low DKK3 expression in neuroblastoma correlated with a poor prognosis. The expression of DKK3 in the tumor series and in neuroblastoma cell lines was inversely correlated with the expression of the MYCN oncogene. Analysis of 2 neuroblastoma cell lines with inducible activity of MYCN showed that DKK3 is down‐regulated by MYCN. We subsequently generated cell lines with inducible expression of DKK3, which revealed an inhibitory effect of DKK3 on proliferation. High DKK3 expression in the benign ganglioneuromas and down‐regulation of DKK3 by MYCN in neuroblastoma might contribute to the strongly different clinical behavior of both neuroblastic tumor types.